The first metazoa living in permanently anoxic conditions

Danovaro, Roberto; Dell'Anno, Antonio; Pusceddu, Antonio; Gambi, Christina; Bang- Berthelsen, Iben Heiner; Kristensen, Reinhardt Møbjerg

Published in: BMC Biology

DOI: 10.1186/1741-7007-8-30

Publication date: 2010

Document version Publisher's PDF, also known as Version of record

Document license: CC BY

Citation for published version (APA): Danovaro, R., Dell'Anno, A., Pusceddu, A., Gambi, C., Bang-Berthelsen, I. H., & Kristensen, R. M. (2010). The first metazoa living in permanently anoxic conditions. BMC Biology, 8, [30]. https://doi.org/10.1186/1741-7007-8- 30

Download date: 28. sep.. 2021 Danovaro et al. BMC Biology 2010, 8:30 http://www.biomedcentral.com/1741-7007/8/30

RESEARCH ARTICLE Open Access TheResearch first article metazoa living in permanently anoxic conditions

Roberto Danovaro*1, Antonio Dell'Anno1, Antonio Pusceddu1, Cristina Gambi1, Iben Heiner2 and Reinhardt Møbjerg Kristensen2

Abstract Background: Several unicellular organisms (prokaryotes and protozoa) can live under permanently anoxic conditions. Although a few metazoans can survive temporarily in the absence of oxygen, it is believed that multi-cellular organisms cannot spend their entire life cycle without free oxygen. Deep seas include some of the most extreme ecosystems on Earth, such as the deep hypersaline anoxic basins of the Mediterranean Sea. These are permanently anoxic systems inhabited by a huge and partly unexplored microbial biodiversity. Results: During the last ten years three oceanographic expeditions were conducted to search for the presence of living fauna in the sediments of the deep anoxic hypersaline L'Atalante basin (Mediterranean Sea). We report here that the sediments of the L'Atalante basin are inhabited by three species of the animal phylum Loricifera (Spinoloricus nov. sp., Rugiloricus nov. sp. and Pliciloricus nov. sp.) new to science. Using radioactive tracers, biochemical analyses, quantitative X-ray microanalysis and infrared spectroscopy, scanning and transmission electron microscopy observations on ultra-sections, we provide evidence that these organisms are metabolically active and show specific adaptations to the extreme conditions of the deep basin, such as the lack of mitochondria, and a large number of hydrogenosome-like organelles, associated with endosymbiotic prokaryotes. Conclusions: This is the first evidence of a metazoan life cycle that is spent entirely in permanently anoxic sediments. Our findings allow us also to conclude that these metazoans live under anoxic conditions through an obligate anaerobic metabolism that is similar to that demonstrated so far only for unicellular eukaryotes. The discovery of these life forms opens new perspectives for the study of metazoan life in habitats lacking molecular oxygen.

Background in the surface centimetre) [4]. These environments are More than 90% of the ocean biosphere is deep (average inhospitable to most marine species [5], except host depth, 3,850 m) and most of this remains unexplored [1]. prokaryotes, protozoa and some metazoans that can tol- The oceans host life at all depths and across the widest erate these environmental conditions [4,6]. Permanently ranges of environmental conditions (that is, temperature, anoxic conditions in the oceans are present in the subsur- salinity, oxygen, pressure), and they represent a huge res- face seafloor [7], and among other areas, in the interior of ervoir of undiscovered biodiversity [2,3]. Deep-sea eco- the Black Sea (at depths >200 m) [8] and in the deep systems also contain the largest hypoxic and anoxic hypersaline anoxic basins (DHABs) of the Mediterranean regions of the Biosphere. The oxygen minimum zones Sea [9,10]. All of these extreme environments are (OMZ) are widely distributed across all of the oceans, at assumed to be exclusively inhabited by viruses [11], Bac- depths generally from 200 m to 1,500 m, and cover teria and Archaea [7-10]. The presence of unicellular approximately 1,150,000 km2. These are characterised by eukaryotes (for example, protozoan ciliates) in anoxic very low oxygen availability (O2 < 0.5 mM) and high sul- marine systems has been documented for decades [12] phide concentrations in the bottom sediments (>0.1 mM and recent findings have indicated that some benthic for- aminifera can be highly adapted to life without oxygen * Correspondence: [email protected] [13]. For limited periods of time, a few metazoan taxa can 1 Department of Marine Science, Faculty of Science, Polytechnic University of tolerate anoxic conditions [6,14]. However, so far, there is Marche, Via Brecce Bianche, 60131 Ancona, Italy no proof of the presence of living metazoans that can © 2010 Danovaro et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Danovaro et al. BMC Biology 2010, 8:30 Page 2 of 10 http://www.biomedcentral.com/1741-7007/8/30

spend their entire life cycle under permanently anoxic to the genera Spinoloricus (Figure 1c, similar to the new conditions [12]. species of Spinoloricus turbatio, which was recently dis- Metazoan meiofauna (multi-cellular organisms of size covered in the deep-sea hydrothermal vents of the Galá- ranging from a few micrometres to 1 mm) [15] represent pagos Spreading Centre) [21], Rugiloricus (belonging to 60% of the metazoa abundance on Earth, and have a long the cauliculus-group; Figure 1e) and Pliciloricus (Figure evolutionary history and high phyletic diversity. They 1f) [22]. include 22 of the 35 animal phyla, six of which are exclu- The permanent reducing conditions of anoxic sedi- sive of the meiofauna (Gnathostomulida, Micrognatho- ments can preserve dead organisms and their protein for zoa, Gastrotricha, Tardigrada, Kinorhyncha, and a long time, so that microscopic analyses do not provide Loricifera, the most recently described animal phylum) proof of the viability of an organism. However, the abun- [16]. These phyla lack larval dispersal in the water col- dance of these loriciferans was the highest reported so far umn and spend their entire life cycle in the sediment. All world-wide per unit of surface sediment investigated of these characteristics make meiofauna the ideal organ- (range: 75 to 701 individuals m-2). This finding is per se ism for investigating metazoan life in systems without surprising, as only two individuals of the phylum Loric- oxygen [17,18]. ifera have been found in the deep Mediterranean Sea over The six DHABs of the Mediterranean Sea are extreme the last 40 years [23-25]. Deep-sea oxygenated sediments environments at depths >3,000 m that have been created in the neighboring of the L'Atalante basin were also inves- by the flooding of ancient evaporites from the Miocene tigated at the time of sampling as well as in several other period (5.5 million years before the present) [19]. Among occasions since 1989, and we never found one single indi- these, the L'Atalante basin displays a 30 to 60 m thick vidual of the phylum Loricifera in the entire Ionian basin. hypersaline brine layer with a density of 1.23 g cm-3 [9], Moreover, the analysis of the oxygenated deep-sea sedi- which represents a physical barrier that hampers oxygen ments surrounding the L'Atalante basin revealed the exchange between the anoxic sediments and the sur- dominance of nematodes and copepods (>95% of the rounding seawaters. This basin is therefore completely total meiofaunal abundance; Additional file 3) and the oxygen free, rich in hydrogen sulphide, and hosts an absence of loriciferans. The density of the Loricifera incredibly diverse and metabolically active prokaryotic extracted from the sediment of the L'Atalante basin assemblages that have adapted to these conditions [9]. In (determined by density gradient) was 1.15 to 1.18 g cm-3, 1998, 2005 and 2008 we carried out three oceanographic whereas the density of the brines above the sediment is expeditions to search for the presence of living fauna in significantly higher (1.23 g cm-3). Moreover, the presence the sediments of the anoxic L'Atalante basin (Additional of laminated sediment layers along with the lack of tur- file 1). bidites in the L'Atalante basin [26] indicates the lack of lateral transport from adjacent systems. These indepen- Results and Discussion dent evidences make very unlikely the sedimentation or In all of the sediments collected from the inner part of the transfer of Loricifera or their carcasses from the oxygen- anoxic basin, we found specimens belonging to three ani- ated sediments surrounding the anoxic basin. mal Phyla: Nematoda, Arthropoda (only Copepoda) and Specimens of the undescribed species of both genera Loricifera. The presence of metazoan meiofauna under Spinoloricus and Rugiloricus had a large oocyte in their permanently anoxic conditions has been reported previ- ovary, which showed a nucleus containing a nucleolus ously also from the deep-sea sediments of the Black Sea, (Figure 1d, e). This is the first evidence of Loricifera although these records were interpreted as the result of a reproducing in the entire deep Mediterranean basin. rain of cadavers that sunk to the anoxic zone from adja- Microscopic analyses also revealed the presence of empty cent oxygenated areas [20]. Our specimens collected exuviae from moulting loriciferans (Figure 1g), suggesting from the L'Atalante basin were initially stained with a pro- that these metazoans did grow in this system. Moreover, tein-binding stain (Rose Bengal) and examined under the scanning electron microscopy confirmed the perfect microscope; here, all of the copepods were empty exu- integrity of these loriciferans (Figure 2), while all of the viae, and the nematodes were only weakly stained (sug- other meiofaunal taxa were largely damaged or degraded. gesting that they had been dead for a while, Figure 1a, b), A second expedition was dedicated to the demonstra- whereas all of the loriciferans, if stained, were intensely tion of the viability of these loriciferans of the L'Atalante coloured (Figure 1c, d). Differences in the colour intensity basin, through independent experimental approaches. All between live and dead metazoans were confirmed by of the experiments were conducted on deck (101,325 Pa), additional experiments on deep-sea nematodes and cope- under anoxic conditions (in a N2 atmosphere), in the dark pods (Additional file 2). The taxonomic analysis revealed and at the in-situ temperature (ca 14°C) immediately after that the loriciferans collected in the anoxic sediments sample retrieval. In the first investigations, intact and belong to three species that are new to science and belong Danovaro et al. BMC Biology 2010, 8:30 Page 3 of 10 http://www.biomedcentral.com/1741-7007/8/30

a b C

d e Oocyte

Oocyte

g f

Figure 1 Metazoans retrieved from the deep hypersaline anoxic L'Atalante basin. (a) Light microscopy (LM) image of a Copepod exuvium (stained with Rose Bengal); (b) LM image of dead nematode (stained with Rose Bengal); (c) LM image of the undescribed species of Spinoloricus (Lo- ricifera; stained with Rose Bengal); (d) LM image of the undescribed species of Spinoloricus stained with Rose Bengal showing the presence of an oo- cyte; (e) LM image of the undescribed species of Rugiloricus (Loricifera, stained with Rose Bengal) with an oocyte; (f) LM image of the undescribed species of Pliciloricus (Loricifera, non stained with Rose Bengal); (g) LM image of moulting exuvium of the undescribed species of Spinoloricus. Note the strong staining of the internal structures in the stained loriciferans (c and d) vs. the pale colouration of the copepod and nematode (a, b). The loriciferan illustrated in Figure 1e was repeatedly washed to highlight the presence of the internal oocyte. Scale bars, 50 μm. undisturbed sediment cores were injected with (3H)-leu- todes. Thanks to this experiment we demonstrated the cine (Table 1) to investigate the ability of these loricifer- presence of significant differences in the incorporation of ans to take up this radiolabelled amino acid. Following radio-labelled compounds and proved the linearity multiple and replicated incubations and controls (that is, between the number of nematodes and the incorporated loriciferans killed before radiolabelled substrate injec- radioactivity (Table 1). These results, per se, are sufficient tion), it was revealed that over a short time scale (four to provide compelling evidence of the activity of the hours), the loriciferans incorporated amounts of this organism from the anoxic systems, but we further investi- radioactive substrate that were significantly higher than gated the viability of the Loricifera collected from the in the controls (that is, killed loriciferans). Decompres- L'Atalante basin by incubating intact and undisturbed sion can alter significantly metabolic activities of deep- sediment cores containing the loriciferans with 5-chlo- sea organisms during their recovery. However, in our romethylfluorescein diacetate (Cell-Tracker™ Green, experiments this potential bias was the same for both the CMFDA: Molecular Probes, Inc., Eugene, Oregon, US) controls and the samples containing live Loricifera. which has been previously used to identify living unicel- Moreover, the ultra-structural analyses did not show any lular eukaryotes in anoxic sediments [6]. This fluorogenic evidence of cell lysis related to the decompression. To test probe labels hydrolytically active (that is, live) cells [6]. the reliability of the approach utilized we sampled living Comparative analyses conducted on anoxic sediments by nematodes from oxygenated sediments and made incuba- confocal laser microscopy on Loricifera kept alive and tions with (3H)-leucine of both living and killed nema- others that were killed prior to incubation revealed, on Danovaro et al. BMC Biology 2010, 8:30 Page 4 of 10 http://www.biomedcentral.com/1741-7007/8/30

average, 40% higher fluorescence intensity in the living Loricifera than in recently killed specimens and the intense fluorescence increased from the outer to the inner parts of the organism (Figure 3a, b). The treatment for the preparation of the controls (that is, Loricifera killed prior to incubation by deep freezing) did not inhibit completely the enzymatic activities present in the body of the animals and therefore we expected the presence of some fluorescence also in the body of the pre-killed ani- mals. This effect has been tested also on different species of living nematodes collected from oxygenated sediments by means of repeated (n = 5) incubation experiments with CellTracker™ Green CMFDA. The differences between living and recently killed nematodes analyzed by confocal laser microscopy were in the same order of the differ- ences encountered between alive and recently killed Loricifera. All of these findings provide the first evidence that the anoxic sediments of the L'Atalante basin are colonised by natural populations of loriciferans, and that these meta- zoans are metabolically active and able to reproduce. The adaptations to permanently anoxic conditions associated with high density/salinity and high hydrogen

Figure 2 Morphological details of the undescribed species of Spinoloricus (Loricifera). Scanning electron microscopy (SEM) image Figure 3 Incorporation of Cell-Tracker™ Green CMFDA by loric- of (a) ventral side of a whole animal with the introvert out (note the lo- iferans from the anoxic sediments of the L'Atalante basin. Series of ricated abdomen with eight plates); (b-c) anterior edge of the lorica confocal laser microscopy images across different sections of the body showing the genus character of the genus Spinoloricus (additional volume of the loriciferans. Sections 1-21 represent the progressive spikes); and (d) posterior lorica with honey-comb structure. No scanning of the loriciferans (undescribed species of Spinoloricus) from prokaryotes are evident on the surface of the bodies of the loriciferans. the outer to the inner part of the body. (a) Cell-Tracker™ Green CMFDA Scale bars, as indicated. treated loriciferans; and (b) Loriciferans killed by freezing prior to Cell- Tracker™ Green CMFDA treatment and used as a control. Danovaro et al. BMC Biology 2010, 8:30 Page 5 of 10 http://www.biomedcentral.com/1741-7007/8/30

Table 1: Radiolabelled substrate incorporation in loriciferans from the L'Atalante basin and nematodes from coastal Mediterranean sediments.

DPM ind-1 Control (killed) Treated (living)

Loriciferans 53 ± 3 93 ± 4

Nematodes (n = 1 ind) 56 ± 1 64 ± 3 (n = 2 ind) 58 ± 4 73 ± 7 (n = 5 ind) 61 ± 1 79 ± 4 (n = 10 ind) 62 ± 1 98 ± 2 Reported are individual disintegrations per minute of nematodes (coastal Mediterranean Sea) and loriciferans (L'Atalante basin) incubated with (3H)-leucine. Data are means of replicates (n = 3-5) ± standard deviation determined on living specimens (treatment) and Loricifera collected from same anoxic sediments but killed prior to treatment (control). Ind: individuals; DPM: disintegration per minute.

sulphide concentrations imply that these organisms have replaced by hydrogenosome-like organelles (Figure 4a, b, developed specific mechanisms for: (i) tolerating an enor- c). The hydrogenosome-like structures showed a perfect mous osmotic pressure (due to the high salinity and integrity of their membranes as well as the presence of a hydrostatic pressure); (ii) detoxifying highly toxic com- marginal plate (Figure 4b). These organelles have been pounds (due to the high hydrogen sulphide concentra- previously encountered in various unrelated unicellular tions); and (iii) living without oxygen. Quantitative X-ray eukaryotes [27,28], but have never been observed so far micro-analysis and Fourier-transformed infrared spec- in multi-cellular organisms (including the facultative troscopy on the body composition of the loriciferans col- anaerobes that face extended periods of aerobiosis during lected from the anoxic sediments revealed significant their life cycle) [14]. Moreover, the Loricifera retrieved differences with the loriciferans collected in the oxygen- from anoxic sediments contained hydrogenosome fields ated deep Atlantic Ocean (Additional files 4, 5 and 6). (Figure 4c) similar to those reported in anaerobic ciliates Loriciferans from the L'Atalante basin had a Ca content [29,30]. Previous studies have reported the ability of (expressed as percentage) that was nine-fold lower than multi-cellular organisms to survive in oxygen-free envi- in specimens inhabiting oxygenated sediments, on aver- ronments, but only for limited periods of time or for a age, and showed Mg, Br and Fe, which were absent in the part of their life cycle [14]. The very high abundance of loriciferans from oxygenated sediments. Moreover, loric- hydrogenosomes within the Loricifera of the L'Atalante iferans from both oxic and anoxic sediments had similar basin and the presence of hydrogenosomes fields repre- concentrations of Na and S, in spite of the much higher sent the first discovery for multicellular organisms. Since salinity and sulphide concentration present in the deep- the hydrogenosomes do not coexist with mitochondria anoxic sediments of the L'Atalante basin (Additional files and they are present only in obligate anaerobic eukary- 4 and 6). Moreover, Fourier-transformed infrared spec- otes (type II anaerobes) [31], these data exclude the possi- troscopy analyses indicated that the lorica of the loricifer- bility that the Loricifera encountered in the anoxic basin ans inhabiting oxygenated deep-sea sediments was are carcasses of organisms inhabiting oxygenated sedi- apparently made of chitin, which was replaced by a chitin ments and transported/sedimented into the anoxic basin. derivative, similar to chitosan, in the loriciferans inhabit- Moreover, the transmission electron microscopy also ing anoxic sediments (Additional file 6). These results revealed the presence of rod-shaped structures (Figure suggest the presence of chemical/structural adaptations 4d, e, f), likely prokaryotes, in close proximity to the of these loriciferans that can inhabit these anoxic sedi- hydrogenosome-like organelles (Figure 4d). These struc- ments of the L'Atalante basin. Scanning electron micros- tures and their spatial distribution resemble the associa- copy revealed the lack of prokaryotes attached to the tion between hydrogenosomes and methanogenic body surface of the loriciferans (Figure 2). Ultra-struc- Archaea, documented so far only in protozoans living in tural analyses carried out by transmission electron permanently anoxic conditions [29,30]. microscopy revealed the lack of mitochondria, which are Danovaro et al. BMC Biology 2010, 8:30 Page 6 of 10 http://www.biomedcentral.com/1741-7007/8/30

Methods Study area and sampling D E The L'Atalante deep hypersaline anoxic basin (DHAB) was discovered in the Mediterranean Sea in 1993 during an expedition that was a part of the European funded project "Mediterranean Ridge Fluid Flow". The bottom of + + the L'Atalante basin is a relatively flat area bounded to the southwest by the Cleft Basin and it is characterised by a morphological escarpment that is several hundreds of P metres high, which is the sea-bottom expression of the main back thrust of the accretionary ridge. These charac- teristics originated from the dissolution of buried salt deposits (evaporitic deposits), which remained from the F + G hypersaline waters of the Miocene period (5.5 My before + present). The L'Atalante basin is characterised by the + presence of a thick brine layer (ca. 40 m) with high den- + sity (1.23 g cm-3) and high contents of Na+ (4,674 mM), + 3 Cl- (5,289 mM) and Mg+ (410 mM) [9]. This layer limits + + the mixing with the overlying oxic deep-waters to only the upper 1 m to 3 m of the brine, and it additionally acts + as a physical barrier for particles settling to the bottom + + sediments. As a result, the inner part of the L'Atalante + basin is completely anoxic since 53,000 yrs before present [32] and is characterized by elevated methane (0.52 mM) H I and hydrogen sulphide (2.9 mM) concentrations [9]. 3 Undisturbed sediment samples (down to a depth of 30 cm) were collected using a USNEL type box corer (sur- face ca. 0.2 m2), in 1998, 2005, 2006 and 2008. The sam- 3 ples from the DHAB sediment were collected in 3 December 1998 (at 3,363 m depth, 35°18.20'N, 21°23.33'E), August 2005 (at 3,600 m depth, 35°18.23'N, 21°23.33'E), and June 2008 (at 3,450 m depth, 35°18.18'N, 21°23.35'E). In 1998 and 2008 additional sediment sam- Figure 4 Electron micrographs of the internal body of loriciferans ples were collected outside the L'Atalante basin (ca. 10 from the deep hypersaline anoxic L'Atalante basin. Illustrated are: miles from the DHAB; 35°11.84'N, 21°24.75'E) at ca. 3,250 (a) a hydrogenosome-like organelle; (b) hydrogenosome-like organ- m depth, for investigation of the characteristics of meio- elle with evidence of the marginal plate; (c) a field of hydrogenosome- like organelles; (d) the proximity between a possible endosymbiotic faunal metazoans from the oxygenated adjacent systems prokaryote and hydrogenosome-like organelles; (e-f) the presence of (three sampling sites per period with three to five repli- possible endosymbiotic prokaryotes; H = Hydrogenosome-like organ- cated deployments per site). In the northern-eastern elles, P = possible endosymbiotic prokaryotes, m = marginal plate. Atlantic Ocean, oxygenated deep-sea sediment samples Scale bars, 0.2 μm. (55°29.87'N, 15°48.61'W at 600 m depth) were collected during the 2006 expedition. Loriciferans retrieved from Conclusions these sediments were used for the comparison of their The results reported here support the hypothesis that the body composition with loriciferan specimens collected in loriciferans inhabiting the anoxic sediments of the L'Atal- the anoxic sediments of the L'Atalante basin. Sediments ante basin have developed an obligate anaerobic metabo- retrieved from the deep anoxic basin were immediately lism and specific adaptations to live without oxygen. processed under strict anaerobic conditions. Although the evolutionary/adaptative mechanisms lead- ing to the colonisation of such extreme environments by Extraction and identification of benthic metazoans these metazoans remain an enigma, this discovery opens For the extraction of metazoan fauna from the sediments, new perspectives for the study of metazoan life in habi- the samples (top 15 to 20 cm of the sediment cores) were tats lacking molecular oxygen. pre-filtered through a 1,000-μm mesh (to remove larger debris), and a 20-μm mesh was used to retain all of the multi-cellular organisms. The fraction remaining on this Danovaro et al. BMC Biology 2010, 8:30 Page 7 of 10 http://www.biomedcentral.com/1741-7007/8/30

latter sieve was re-suspended and centrifuged three times ture, the sediments were incubated with an aqueous solu- with Ludox HS40 (density 1.31 g cm-3) [33]. All of the tion of (3H)-leucine and then processed as described organisms isolated were counted and classified according above. We used deep freezing to kill animals, as previous to standard protocols [34,35]. Only the organisms col- studies have demonstrated that meiofauna fixed using lected during the first expedition were stained with Rose chemical compounds (that is, formaldehyde, glutaralde- Bengal (0.5 g L-1), a stain commonly used to highlight the hyde and ethanol) show a significant loss in the incorpo- body structures under light microscopy. On average of all rated radioactivity [35]. All samples were incubated on - collected samples metazoan abundance was 2,075 ind. m deck (101,325 Pa) under anoxic conditions (N2 atmo- 2 in the L'Atalante sediments vs 21,548 ind. m-2 in the oxy- sphere) for four hours in the dark, and at the in-situ tem- genated sediments surrounding the basin. In the anoxic perature (about 14°C). At the end of the incubations, the sediments of the L'Atalante basin, Loricifera accounted samples were deep-frozen in liquid N2 to stop any addi- for 16.1% of the total metazoan abundance. No Loricifera tional substrate uptake. In the laboratory, the organisms were encountered in the oxygenated sediments surround- were extracted from the sediment as previously ing the basin, where nematodes and copepods accounted described. Due to the relatively low numbers of loricifer- for 95% and 4%, respectively, of the total metazoan abun- ans in the sediment cores (n = 3 both in the control and dance. treated samples) the organisms were analyzed individu- ally. Meiofaunal organisms were rinsed with 0.2-μm pre- Identification of loriciferans to genus and species level with filtered seawater (to minimise interference due to radio- light and scanning electron microscopy activity incorporated by prokaryotes that were potentially The extracted specimens were mounted on microslides in present on the metazoan surface) [37] and transferred to a drop of distilled water. The water was progressively scintillation vials. The samples were digested at 50°C for replaced by increasing glycerol concentrations (5%, 10%, 24 h using 1 mL tissue solubiliser (Soluene-350, Packard 25%, 50% and 100% vol water:vol glycerol). Then the Inc., Meriden, Connecticut, US). After addition of 10 mL specimens were sealed with Glyceel. The microslides scintillation cocktail, the radioactivity (as disintegration were analyzed using a light microscope with phase con- per minute; DPM) in the loriciferans was determined in a trast and Nomarski DIC optics. Micrographs of the speci- liquid scintillation counter (Packard, Tri-Carb 2100 TR). mens were taken on an Olympus BX51 microscope DPM data were normalised per individual. equipped with a digital Olympus C-3030 zoom camera To test the accuracy and consistency of the radiotracer and on a Leica DMRXA microscope with a digital Leica experiments carried out on sediments collected in the DC200 camera (Leica Camera AG, Solms, Germany). L'Atalante basin, additional experiments were performed Morphological details of the loriciferans were obtained on coastal sediments of the Mediterranean Sea. Lorifer- by scanning electron microscopy. Loriciferans extracted ans were not present in these samples; therefore nema- from sediments were carefully rinsed in distilled water todes were used as model organisms. After incubation and then dehydrated through a graded series of ethanol with the radiolabelled substrate, the nematodes (diame- and acetone prior to critical-point drying. The dried ter: 20 to 30 μm and length: 200 to 900 μm) were specimens were mounted on aluminium stubs and coated extracted from the sediments and analyzed individually with gold prior to observation under scanning electron or pooled together (from 2 to 10 individuals). These microscopy (Philips XL20, Philips Electronics, Eind- experiments demonstrated that the radioactivity incorpo- hoven, The Netherlands). rated into the nematodes is significantly higher than that Incubation experiments found in organisms used as controls, even when a single 3 Incorporation of ( H)-leucine For investigating the individual is analyzed (Table 1). Moreover, radioactivity vitality of the meiofaunal metazoans, the top 5 cm of measured from the nematodes incubated with radioac- 3 intact sediment cores were incubated with ( H)-leucine tive substrates increased linearly with the increasing [36]. Replicate sediment samples (n = 3, internal diameter number of individuals analyzed. 5.5 cm, approximately 120 cm3 of sediment per replicate Incorporation of Cell-Tracker™ Green CMFDA After sample) were kept in the dark at in-situ temperature and sediment retrieval from the anoxic basin, the top 5 cm of under anoxic conditions (a N2 atmosphere); these were the sediment cores and its anoxic overlying water were 3 injected with 10 mL ( H)-leucine dissolved in 0.2 μm fil- maintained under strict anaerobic conditions (N2 atmo- tered, autoclaved and degassed deep-sea water (final con- sphere) and incubated on deck (101,325 Pa) in the dark centration 0.2 mCi mL-1). Controls for the incubation and at the in-situ temperature (ca 14°C). The samples experiments were obtained as follows: additional sedi- were used for incorporation experiments with Cell- ment cores were frozen immediately after collection at - Tracker™ Green CMFDA, fluorescent probe (5-chlorom- 80°C, to kill all metazoans within the samples. After ethylfluorescein diacetate; Molecular Probes, Inc., thawing, when the samples reached the in-situ tempera- Danovaro et al. BMC Biology 2010, 8:30 Page 8 of 10 http://www.biomedcentral.com/1741-7007/8/30

Eugene, Oregon, US; 10 μM final concentration). The the posterior lorica and the whole organism (Additional Cell-Tracker™ Green fluorescent CMFDA probe pene- file 4). trates the cells and reacts with the intracellular enzymes, Spectroscopic infra-red determinations generating fluorescence [38]. This molecular probe is Fourier transformed Infra-Red (FT-IR) spectroscopic specifically designed for testing the presence of metabolic determinations were carried out on loriciferans collected activity and is therefore used here to support the evi- both from the anoxic sediments of the L'Atalante basin dence of viability of the metazoans present within the and from oxic sediments of the NE Atlantic Ocean. Spec- anoxic deep-sea sediments. The sediment samples were tral data were obtained with a Perkin-Elmer Spectrum incubated for four hours. Controls for the incubation One FT-IR equipped with a Perkin-Elmer Autoimage experiments were obtained as follows: additional sedi- microscope (PerkinElmer Life and Analytical Sciences, ment cores were frozen immediately after collection at - Shelton, Connecticut, US). Spectra were measured from 80°C to kill all metazoans within the samples. After thaw- 4,000 to 400 cm-1 at a spectral resolution of 4 cm-1 with ing, when the samples reached the in-situ temperature, 128 scans. The spatial resolution was 30 × 30 μm. Back- the sediments were incubated with an aqueous solution ground scans were obtained from a region of no sample Cell-Tracker™ Green CMFDA, and then processed as and rationed against the sample spectrum. The samples described above. At the end of the incubation, the sam- were deposited first on a steel support to collect reflec- ples were deep-frozen in liquid N2 to stop any metabolic tance spectra and on the centre of a BaF2 plate for trans- reactions, and the recovered loriciferans were placed on mittance spectral acquisition. Specific areas of interest concave slides containing a drop of 0.9% NaCl solution were identified by means of the microscope television (previously autoclaved). The fluorescence of the organ- camera. Baseline (polynomial line fit) was performed in isms was examined using a confocal microscope all cases while Second Derivative, Fourier Self Deconvo- equipped with Kr/Ar mixed gas laser (Bio-Rad MRC 1024 lution and Curve Fitting (Gaussian character) procedures UV; Bio-Rad, Hercules, California, US) using excitation were used to determine the absorbance ratio between the wavelengths 488 nm and the emission has been detected bands of interest. All spectra were scaled for equal inten- after passing a bandpass filter of 522/35 nm. The confocal sity in the Amide I band. For data handling, the Spectrum laser images were acquired (using the same laser emission v.303 (Perkin-Elmer) software package was used. power, iris and electronic gain for all acquisitions) in the Analysis of the ultra-structure of loriciferans by transmission Bio-Rad PIC format using the Bio-Rad Lasersharp Acqui- electron microscopy sition software (Release 2.1). The organisms were investi- For ultrastructural studies, loriciferans (undescribed spe- gated using exactly the same magnification (× 40) in cies of the genus Rugiloricus) extracted from sediments order to allow data comparison. Images were taken at were carefully rinsed in distilled water and then stored in depths of 3 μm for a total of 21 sections per animal and glutaraldehyde (2% final solution) for transmission elec- analyzed using the Bio-Rad Lasersharp processing tool. tron microscopy examinations. After treatment with This enabled merging all of the sections (without any osmium (one hour incubation) and acetone dehydration contrast manipulation) and measuring the mean scale (two times at 60% for one minute, and three times at colour (0 to 255) of the entire animal body. Images were 100% for one minute), loriciferans were embedded in sequentially acquired and stored as TIFF files. The reli- epoxy resin. Ultrathin sections (78 nm) were obtained ability of the control used in the experiment was previ- using a microtome (Model RMC MTX, Boeckeler Instru- ously tested by means of repeated (n = 5) incubation ments Inc., Tucson, Arizona, USA) equipped with a dia- experiments with Cell-Tracker™ Green CMFDA per- mond knife. Sections were collected on carbon-coated formed on two nematode species cultured in the labora- formvar supports, stained with lead citrate, and examined tory (Diplolamelloides myily and Diplolaimella by transmission electron microscopy (Philips EM 208). diewgatentis). All of the specimens were analyzed by con- focal laser microscopy, as described above. Additional material X-ray micro-analysis of the elemental composition of Loricifera Additional file 1 The study area. Location of the sampling areas in the After extraction from the sediment, loriciferans from central Mediterranean Sea, showing: (a), area (rectangle) including the deep hypersaline anoxic basin (x axis: Longitude; y axis: Latitude); and (b), both the L'Atalante basin (undescribed species of the detailed contour map of the L'Atalante basin. genus Spinoloricus, only adults) and the deep NE Atlantic Ocean (Rugiloricus cauliculus cfr) underwent quantita- tive X-ray micro-analysis, after coating with graphite. Specimens collected in the oxygenated sediments were used as a reference. The selected parts were: abdomen, Danovaro et al. BMC Biology 2010, 8:30 Page 9 of 10 http://www.biomedcentral.com/1741-7007/8/30

Author Details Additional file 2 The effect of Rose Bengal on living and dead speci- 1Department of Marine Science, Faculty of Science, Polytechnic University of mens. (a and b) Light microscopy (LM) images of living deep-sea nema- Marche, Via Brecce Bianche, 60131 Ancona, Italy and 2Natural History Museum todes collected from oxygenated sediments adjacent to the anoxic basin of Denmark, Zoological Museum, Invertebrate Department, Universitetsparken and stained with Rose Bengal; (c) LM image of dead deep-sea nematode 15, DK-2100 Copenhagen, Denmark stained with Rose Bengal; (d) LM image of living deep-sea copepods col- lected from oxygenated sediments adjacent to the anoxic basin and Received: 11 January 2010 Accepted: 6 April 2010 stained with Rose Bengal; (e) LM image of deep-sea copepod exuviae Published: 6 April 2010 stained with Rose Bengal. 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doi: 10.1186/1741-7007-8-30 Cite this article as: Danovaro et al., The first metazoa living in permanently anoxic conditions BMC Biology 2010, 8:30